Abstract Study of the duplicated glycolytic genes in Lactococcus lactis IL1403.
The conversion of sugars into lactic acid is the main metabolic pathway providing energy to lactic acid
bacteria. This conversion is also involved in production of different compounds participating to the
organoleptic properties of fermented products. The L. lactis knowledge of the genome has given the
access to sequences of genes encoding the enzymes involved in the two main metabolic pathways described
for the fermentation of glucose in lactic acid bacteria: (1) the homofermentative pathway through
glycolysis leading to two lactate molecules per glucose consumed; (2) the heterofermentative pathway
through the Pentose Phosphate pathway giving one lactate, one acetate and one CO2 per molecule of
glucose. The research of the genes, corresponding to proteins involved in these metabolic pathways,
revealed that some enzymes are encoded by 2 distinct genes. This fact could give to the cell the
possibility to produce enzymes with different biochemical properties, or to control their expression
according to specific conditions. two copies of genes potentially encoding glyceraldehyde-3-phosphate
dehydrogenase (gap) and enolase (eno) have been identified. Other microorganisms such as
E. coli and B. subtilis also possess 2 gap genes sharing up to 60% homology, but having
different functions. In L. lactis, gap1 and gap2 genes share around 80% identity at
both the nucleotidic and protein level. The analysis of codon usage, the transcription and the effect of
genes inactivation shows that gap1 is the only gene involved in glycolysis. The transcription of
this essential gene is very high during all phases of growth. Low increase of the level of transcription
could be evidenced during growth in glucose, a sugar inducing the Catabolite Repression. Moreover, the
presence of potential fixation site for CcpA (Cre box) upstream of initiation transcription box -35
suggests that gap1 transcription is activated by this protein. In contrast, the gap2 gene is
dispensable and expressed at a very low level in our experimental conditions. Finally, in opposition to
GapB from B. subtilis, the product of L. lactis gap2 might not to be involved in the
neoglucogenesis. enoA and enoB genes are coding for proteins sharing 55% identity with known
enolase. In opposition to the gap genes, the eno genes does not share significant nucleotidic homologies
together. However, enoA presents 87% identity with the enolase genes from sequenced
Streptococcus species whereas enoB presents 95% identity with a plasmidic encoded gene
isolated from Streptococcus thermophilus. These observations suggest that enoB was transferred
from species to other. The analysis of codons bias strongly suggests that EnoA is the main glycolytic
enolase. The transcription of these two genes is high during the exponential growth, 2 folds higher in
glucose for enoA and similar during glucose or galactose fermentation for enoB. enoA and
enoB seem transcribed simultaneously during the growth. These results suggest that both genes may
play a significant role in the glycolysis.